Streptococcus pneumoniae asymptomatically colonizes the upper respiratory tract of healthy young children and is a leading cause of acute otitis media (OM). S. pneumoniae colonization is a critical step in the disease process and occurs in the context of a complex microbial community. Limited understanding of the relationships between bacterial colonization patterns and risk of OM has hindered the development of preventive and therapeutic strategies that maximize clinical outcomes and minimize disruptions to the protective commensal flora. Our proposed studies are in direct response to the NIDCD Program Announcement (PA-08-092) Novel Approaches to Study Polymicrobial Diseases. We will analyze pre-existing data from a cross sectional study of 249 Philadelphia children 6 months to =3 years of age. Samples and data were collected during the 2009-10 winter respiratory virus season. High throughput 454 16S rRNA gene pyrosequencing will be used to characterize nasal microbial communities. RNA-sequencing (RNA-seq) on the Illumina Genome Analyzer 2 platform will be used to compare S. pneumoniae transcriptional profiles under a range of experimental conditions. Our specific aims are: 1) To characterize upper respiratory tract microbial community structure in a cross sectional sample of healthy children, children with acute OM, and children with other, primarily non-bacterial upper respiratory tract infections, which often precede the onset of acute OM and 2) To develop RNA- seq protocols and bioinformatics pipelines to study S. pneumoniae transcriptional profiles, alone and in the presence of key commensal species. These studies will identify members of the normal flora involved in the causal pathogenic pathway of S. pneumoniae colonization and acute OM. New methods will be developed to identify S. pneumoniae virulence genes uniquely expressed in polymicrobial settings. Over the long term these data will aid in the development of biotherapeutics such as probiotics and will also provide information relevant for the control of other bacterial pathogens that colonize the respiratory tract.